Abstract /Summary Streptococcus pneumoniae is a major global bacterial human pathogen, causing ~1 million deaths annually worldwide, due to pneumonia, sepsis, and meningitis. Two strategies are used to combat such infections. Antibiotics can often cure such infections, and vaccines are used to reduce the circulating populations of the most dangerous serotypes. However, both strategies are failing at an increasing rate. Antibiotic resistant strains are continually arising and spreading globally; vaccination effectiveness is also under challenge, as serotypes not targeted by current vaccine formulations are continually arising and rapidly replace the targeted ones. The cause of these failures is transfer of multiple foreign genes into the bacteria, but the mechanisms creating the new infectious and resistant strain types are unclear. Transfer events are of two types, named as micro- and macro-recombination events. The micro events, involving dozens of hundreds of base pairs, are consistent with the known properties of gene transfer by transformation in pneumococcus. However, the more numerous, and more significant, events involve transfer of multiple blocks of tens of thousands of nucleotides, sometimes all from a single donor strain. These macro-recombination events are difficult to reconcile completely with any known mechanism of gene transfer - whether conjugation, transduction, or transformation. This exploratory project would use microfluidics to create numerous small chambers (droplets) within which attacker-target interactions can be studied and characterized for the first time at both the cellular and molecular levels, by both identifying the participant cells and tracing all gene exchange events at full genome resolution. Medical Relevance. Most pathogenic streptococci share the mechanism of gene transfer by natural genetic transformation. Genetic transformation is an important path for genetic flexibility in pneumococcus, where it is documented as key to vaccine escape and creation and spread of drug-resistance genes. Because Streptococcus pneumoniae is a model organism for the study of DNA uptake, this work on the mechanism that transfers unexpectedly large blocks of genes between strain or species will have broad impact on understanding and targeting many similar peptide regulated gene exchange systems among Gram positive bacteria that are often associated with the ability of these bacteria to cause disease.